Figure 3. CXCR3/CXCL10-mediated brain infiltration is important for the neuroprotective effect of CD8⁺ TRLs.

(A) RT-qPCR of CXCR3 ligand (Cxcl9, Cxcl10, and Cxcl11) expression in brain 1 and 3 days after tMCAO versus sham. n = 3/group. Two-tailed Student’s t test. (B) Expression of CXCL10 was assessed in the blood (left) and in the ischemic brain (right) by ELISA 1 day after tMCAO. n = 5–6/group. Two-tailed Student’s t test. (C) Expression of CXCL10, CXCL11, and CXCL9 was assessed in the CD31⁺ endothelium of the ischemic brain 3 days after stroke. Scale bars: 10 μm. (D–F) Sixty-minute tMCAO was induced in WT, Cxcl10-KO, or Cxcr3-KO mice (D). Brain infiltration by CD8⁺CD122⁺ TRLs was detected by flow cytometry in Cxcl10-KO (E, n = 6) or Cxcr3-KO (F, n = 3) mice 1 day after stroke. PB=pacific blue. Two-tailed Student’s t test. (G and H) CD8⁺ TRLs were sorted from the spleen of WT or Cxcr3^–/– mice and labeled with CFSE (1 μM). Labeled cells were injected (1 × 10⁶/mouse, i.v.) into anti-CD122 mAb–pretreated mice at 2 hours after 60-minute tMCAO. (G) Flow cytometry showed reduced brain infiltration by Cxcr3^–/– CFSE⁺CD8⁺ TRLs 3 days after stroke. The plots are representative of 3 animals in each group. (H) Infarct volumes 3 days after tMCAO. n = 6–8/group. One-way ANOVA and post hoc Bonferroni’s test. (I) The immunomodulatory effect of Cxcr3^–/– CD8⁺ TRLs was intact compared with WT CD8⁺ TRLs. CD3⁺CD25^–CD122^– Teffs were sorted from the spleen of healthy donor mice and cocultured in vitro with CFSE-labeled Cxcr3^–/– or WT CD8⁺ TRLs. Cells were stimulated with PMA (80 nM) and ionomycin (1 μM) for 5 hours. The production of TNF-α and IL-4 in CFSE^–CD3⁺ Teff cells was detected by flow cytometry. n = 3/group. One-way ANOVA and post hoc Bonferroni’s test. *P < 0.05; **P < 0.01; ***P < 0.001.